Inhibitor of apoptosis proteins and nucleic acids and methods for making and using them

ABSTRACT

The invention provides polypeptides comprising inhibitor of apoptosis protein (IAP) family members, such as BmIAP initially derived from  Bombyx mori  BmN cells, and nucleic acids encoding them, and methods for making and using these compositions, including their use for inhibiting apoptosis.

This application claims priority to U.S. Provisional Application Ser.No. 60/260,478, filed Jan. 8, 2001.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made in part with Government support under NationalInstitutes of Health grants ES02701, AG15402, ES 04699, CA30199 (NCI);U.S. Department of Agriculture grants 97-35302-4406, 9802852; BinationalAgriculture Research and Development grant 96-34339-3532; and, NationalInstitute of Environmental Health Science grant ES 05707. The Governmentmay have certain rights in the invention.

TECHNICAL FIELD

This invention generally pertains to the fields of cell biology andmolecular biology. In particular, this invention provides polypeptidescomprising the inhibitor of apoptosis protein (IAP) family member BmIAP,initially derived from silkworm Bombyx mori BmN cells, and nucleic acidsencoding them, and methods for making and using these compositions,including their use for inhibiting caspase proteases and apoptosis.

BACKGROUND

Apoptosis or programmed cell death is a cellular suicide process inwhich damaged or harmful cells are eliminated from multicellularorganisms. Cells undergoing apoptosis have distinct morphologicalchanges including cell shrinkage, membrane blebbing, chromatincondensation, apoptotic body formation and fragmentation. This cellsuicide program is evolutionarily conserved across animal and plantspecies. Apoptosis plays an important role in the development andhomeostasis of metazoans and is also critical in insect embryonicdevelopment and metamorphosis. Furthermore, apoptosis acts as a hostdefense mechanism. For example, virally infected cells are eliminated byapoptosis to limit the propagation of viruses. Apoptosis mechanisms areinvolved in plant reactions to biotic and abiotic insults. Dysregulationof apoptosis has been associated with a variety of human diseasesincluding cancer, neurodegenerative disorders and autoimmune diseases.Accordingly, identification of novel mechanisms to manipulate apoptosisprovides new means to study and manipulate this process.

The first “inhibitor of apoptosis protein” (IAPs) was identified in abaculovirus. The baculovirus IAPs, CpIAP and OpIAP, are able to blockapoptosis induced by p35-deficient baculovirus AcMNPV in insect Sf-21cells. Cellular IAP homologues have been found in various animal speciesincluding worms, insects and humans. IAP proteins have a distinctiveprimary structure. They contain one to three copies of “baculoviral IAPrepeaf” (BIR) domain and most IAPs also contain a RING domain near theirC-termini. The BIR domain contains a highly conserved arrangement ofCys/His residues forming a stable fold that chelates zinc. The BIRregion was also found to interact with regulators of IAPs includingGrim, Reaper and Hid from Drosophila and may also mediatehomo-oligomerization. The RING finger is a common zinc-binding motifthat also exists in other cellular proteins. Recent studies show thatseveral IAPs, including XIAP (a NF-kappaB-dependent member of the IAPgene family, see, e.g., Deveraux (1999) EMBO J. 18:5242-5251), cIAP1,CIAP2, DIAP1, SfIAP and CpIAP are inhibitors of caspases, a family ofintracellular proteases responsible for the execution of the apoptosisprogram. These IAPs can directly bind and inhibit some members ofcaspase family including caspases-3, -7 and -9. Structure-functionstudies have demonstrated that inhibition of caspases-3 and -7 requiresonly a single BIR domain while RING domain may perform other functionsincluding recruitment of ubiquitin conjugating enzymes (UBCs). Whenexpressed without the associated BIRs, the RING region of SfIAP wasfound to enhance the proapoptotic activity of mammalian caspase-9suggesting this domain operates as a trans-dominant inhibitor ofendogenous proteins involved in apoptosis suppression. Ectopicexpression of lepidopteran SfIAP and baculoviral CpIAP blocks apoptosisin mammalian cells, suggesting conservation of the apoptosis programamong various species and a shared mechanism used by the IAP family.

Bombyx mori (silkworm) has been domesticated for silk-production forthousands of years. Used together with baculoviruses, it has also beendeveloped as an organism for large-scale production of foreign proteinsin the biotechnology industry. Despite its extensive use in sericultureand biotechnology, to date no apoptosis-regulating genes of silkwormhave been identified.

SUMMARY

The invention provides an isolated or recombinant nucleic acidcomprising a nucleic acid sequence having at least about 95% sequenceidentity, about 97% sequence identity, about 99% sequence identity toSEQ ID NO:1. In one aspect of the invention, the nucleic acid encodes apolypeptide capable of inhibiting apoptosis in insect cells, encodes apolypeptide capable of inhibiting apoptosis in insect cells, such aslepidopteran and coleopteran cells, e.g., Bombyx mori or Spodopterafrugiperda cells. In one aspect the nucleic acid encodes a polypeptidecapable of inhibiting apoptosis in mammalian cells, encodes apolypeptide capable of inhibiting apoptosis in plant cells, or encodes apolypeptide capable of inhibiting caspase 9, e.g., human caspase 9. Inothers aspects, the isolated or recombinant nucleic acid encodes apolypeptide having a sequence as set forth in SEQ ID NO:2, and,comprises a nucleic acid sequence as set forth in SEQ ID NO:1.

The invention provides an expression cassette (e.g., vector, recombinantvirus) comprising at least one nucleic acid of the invention operablylinked to a promoter. The nucleic acid can comprise a sequence having atleast 95% sequence identity to SEQ ID NO:1. As defined herein, in oneaspect, an expression cassette comprises a nucleic acid of the inventionoperably linked to a promoter. The promoter can be a constitutive or aninducible promoter, or, the promoter can be a developmentally regulatedor a tissue specific promoter. In one aspect, the nucleic acid on theexpression cassette encodes a polypeptide having a sequence as set forthin SEQ ID NO:2.

The invention provides a transformed cell comprising a nucleic acid ofthe invention. This nucleic acid can comprise a sequence having at least95% sequence identity to SEQ ID NO:1. The cell can be a mammalian cell(such as a human cell), an insect cell, such as a Spodoptera frugiperdaor Bombyx mori cell, a plant cell, a bacteria, a yeast cell, and thelike. The transformed cell can comprise a nucleic acid encoding apolypeptide having a sequence as set forth in SEQ ID NO:2. Thesetransformed cells can be used in the screening methods of the invention,which provide for identification of modulators of the polypeptides ofthe invention, or, compositions that specifically bind to thepolypeptides of the invention.

The invention provides a non-human transgenic animal comprising anucleic acid sequence of the invention, e.g., one having at least 95%sequence identity to SEQ ID NO:1. The nonhuman transgenic animal can bea rat or a mouse. The nonhuman transgenic animal can comprise a nucleicacid encoding a polypeptide having a sequence as set forth in SEQ IDNO:2. The nucleic acid can encode a polypeptide capable of inhibitingapoptosis.

The invention provides a transgenic plant comprising a nucleic acidsequence of the invention, e.g., one having at least 95% sequenceidentity to SEQ ID NO:1. The transgenic plant can comprise a nucleicacid encoding a polypeptide capable of inhibiting apoptosis. Thetransgenic plant, as a result of expression of the nucleic acid of theinvention, can become abiotic or biotic insult resistant. The bioticinsult can be induced by a plant pathogen, such as a virus, a fungus, abacteria or a nemotode. The abiotic insult can be induced by highmoisture, low moisture, salinity, nutrient deficiency, air pollution,high temperature, low temperature, soil toxicity, herbicides orinsecticides. The transgenic plant, upon expressing a nucleic acid ofthe invention or being exposed to a polypeptide of the invention, can bephenotypically altered, e.g., wherein at least a portion of the plantexhibits a decreased level of senescence. The invention provides a seedcapable of germinating into a plant having in its genome a heterologousnucleic acid sequence comprising a nucleic acid of the invention, e.g.,one having at least 95% sequence identity to SEQ ID NO:1. The seed cancomprise a nucleic acid encoding a polypeptide capable of inhibitingapoptosis in a plant cell.

The invention provides an isolated or recombinant polypeptide comprisinga sequence having at least 95% sequence identity to SEQ ID NO:2. Theisolated or recombinant polypeptide of the invention can be capable ofinhibiting apoptosis in cells, e.g., in insect cells, such aslepidopteran cells, e.g., Bombyx mori or Spodoptera frugiperda cells,and coleopteran cells, in mammalian cells, in yeast cells, in bacterialcells, in plant cells. In one aspect, the isolated or recombinantpolypeptide of the invention is capable of inhibiting caspase 9. Theinvention provides an isolated or recombinant polypeptide comprising asequence as set forth in SEQ ID NO:2.

The invention provides a fusion protein comprising a polypeptide of theinvention, e.g., a sequence having at least 95% sequence identity to SEQID NO:2, and a second domain. The fusion protein's second domain cancomprise glutathione S-transferase (GST), and other domains, asdescribed below.

The invention provides an antibody or binding fragment thereof, whereinthe antibody or fragment specifically binds to a polypeptide or animmunogenic fragment thereof, wherein the polypeptide comprises asequence having at least 95% sequence identity to SEQ ID NO:2. Theinvention provides an antibody or binding fragment thereof, wherein theantibody or fragment specifically binds to a protein having an aminoacid sequence as set forth in SEQ ID NO:2 or an immunogenic fragmentthereof.

The invention provides an array comprising a nucleic acid comprising anucleic acid of the invention, e.g., a sequence having at least 95%sequence identity to SEQ ID NO:1, or, a fragment thereof.

The invention provides a method of detecting or isolating a polypeptide,wherein the polypeptide comprises a sequence having at least 95%sequence identity to SEQ ID NO:2, comprising contacting a biologicalsample with an antibody as set forth in claim 38 or claim 39. Theinvention provides a method of making a recombinant polypeptidecomprising expressing a nucleic acid comprising a sequence having atleast 95% sequence identity to SEQ ID NO:1.

The invention provides a method for inhibiting apoptosis in a cellcomprising the following steps: (a) providing an isolated or recombinantpolypeptide comprising a sequence having at least 95% sequence identityto SEQ ID NO:2, wherein the polypeptide is capable inhibiting apoptosisin the cell, and, (a) contacting the polypeptide with the cell in anamount sufficient to inhibit apoptosis in the cell. The inventionprovides a method for inhibiting apoptosis in a cell comprising thefollowing steps: (a) providing an isolated or recombinant nucleic acidcomprising a sequence having at least 95% sequence identity to SEQ IDNO:1, wherein the nucleic acid encodes polypeptide capable of inhibitingapoptosis in the cell, and, (b) contacting the nucleic acid with thecell and expressing the nucleic acid to produce an amount of polypeptidesufficient to inhibit apoptosis in the cell. In alternative aspects ofthese methods the cell can be an insect cell, e.g., a lepidopteran cell,such as a Bombyx mori cell or a Spodoptera frugiperda cell, and acoleopteran cell, a mammalian cell, or a plant cell.

The invention provides a method for identifying an agent that canmodulate the activity of a polypeptide, wherein the polypeptidecomprises a sequence having at least 95% sequence identity to SEQ IDNO:2 and is capable inhibiting a caspase 9 protease, comprising: (a)providing an isolated or recombinant polypeptide comprising a sequencehaving at least 95% sequence identity to SEQ ID NO:2 that is capableinhibiting a caspase 9 protease, and a test agent, (b) contacting thecaspase 9 protease and polypeptide in the presence and absence of thetest agent; and, (c) measuring the ability of the polypeptide to inhibitthe caspase 9 protease in the presence and absence of the test agent,wherein an increase or decrease in the ability of the polypeptide toinhibit the caspase 9 protease in the presence of the test agentidentifies the test agent as a modulator of the polypeptide's activity.

The invention provides a method for identifying an agent that canmodulate the activity of a polypeptide, wherein the polypeptidecomprises a sequence having at least 95% sequence identity to SEQ IDNO:2 and is capable inhibiting apoptosis in a cell, comprising: (a)contacting a cell expressing the polypeptide recombinantly in thepresence and absence of a test agent before, during or after inducingapoptosis in the cell; and, (b) measuring the amount or degree of thepolypeptide's activity in the cell in the presence and absence of thetest agent, wherein an increase or decrease in the amount or degree ofapoptosis in the cell in the presence of the test agent identifies thetest agent as a modulator of the polypeptide's activity, In alternativeaspects of these methods the cell can be an insect cell, e.g., alepidopteran cell, such as a Bombyx mori cell or a Spodoptera frugiperdacell, or a coleopteran cell, a mammalian cell, a yeast cell, a bacterialcell, a plant cell, and the like. The degree of the polypeptide'sactivity in the cell can be determined by measuring the amount or degreeof apoptosis in the cell; the amount or degree of caspase proteaseactivity in the cell; the amount or degree of DNA fragmentation in thecell; the amount or degree of cleavage of substrates of caspases in thecell; or by measuring the amount or degree of any surrogate marker ofapoptosis in the cell.

The invention provides a method of generating an abiotic or bioticinsult-resistant plant comprising the following steps: (a) providing anisolated or recombinant polypeptide comprising a sequence having atleast 95% sequence identity to SEQ ID NO:2, wherein the polypeptide iscapable inhibiting apoptosis in a plant cell, and, (a) contacting thepolypeptide with the plant in an amount sufficient to inhibit apoptosisin the plant, thereby generating a plant that is biotic insultresistant. The invention provides a method for generating an abiotic orbiotic insult-resistant plant comprising the following steps: (a)providing an isolated or recombinant nucleic acid comprising a sequencehaving at least 95% sequence identity to SEQ ID NO:1, wherein thenucleic acid encodes polypeptide capable of inhibiting apoptosis in aplant cell, and, (b) contacting the nucleic acid with the plant andexpressing the nucleic acid to produce an amount of polypeptidesufficient to inhibit apoptosis in the plant. In alternative aspects ofthese methods, the biotic insult is induced by a plant pathogen, such asa virus, a fungus, a bacteria or a nemotode. In alternative aspects ofthese methods, the abiotic insult is induced by high moisture, lowmoisture, salinity, nutrient deficiency, air pollution, hightemperature, low temperature, soil toxicity, herbicides or insecticides.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic showing the location of the BIR domains (BIR1,residues 74 to 140; BIR2, residues 182 to 249; of SEQ ID NO:2, as setforth in Example 1, below) and the RING domain (residues 298 to 314; ofSEQ ID NO:2) of BmIAP. FIG. 1B (SEQ ID NO:2) is the full length aminoacid sequence of BmIAP (also set forth in Example 1, below, as SEQ IDNO:2). Sequence alignments of the BIR1 (FIG. 1C) (SEQ ID NOS: 8-13,respectively in order of appearance), BIR2 (FIG. 1D) (SEQ ID NOS:14-19,respectively in order of appearance) and RING (FIG. 1E) (SEQ ID NOS20-25, respectively in order of appearance) domains of BmIAP with thecorresponding domains of other IAP family members are shown. Bold textindicates identical amino acid. See Example 1, below.

FIG. 2 is a representation of photographs of Sf-21 cells taken threedays post-transfection at 40× magnification; the cells wereco-transfected with p35-deficient AcMNPV viral DNA and (FIG. 2A) BmIAP,or (FIG. 2D) SfIAP, (the production of occlusion bodies is indicated byarrowheads); (FIG. 2B) BmIAP-BIR, (FIG. 2C) BmIAP-RING; and, (FIG. 2E)AcIAP; in 2B, 2C and 2E the apoptotic body formation (without occlusionbody formation) are indicated by arrowheads. FIG. 2F depicts controluninfected SF-21 cells. Full details in Example 1, below.

FIG. 3 schematically summarizes the results of experiments showing thatrecombinantly expressed BmIAP protects mammalian cells againstBax-induced but not Fas-induced apoptosis. Expression plasmid encodingBax (FIG. 3A) or Fas (FIG. 3B) were co-transfected into HEK 293 cellswith various myc-tagged IAP expression plasmids. In FIG. 3C recombinantBmIAP was added to cytosolic extracts from HEK293 cells concurrentlywith the addition of cytochrome-c and dATP. Full details in Example 1,below.

FIG. 4 schematically summarizes the results of experiments showing thatrecombinant BmIAP directly suppresses caspase-9 but not caspase-3 orcaspase-7. FIG. 4A: Recombinant active caspase-9 was incubated withAc-LEHD-AFC substrate in the presence or absence of variousconcentration of recombinant purified BmIAP or SfIAP. FIG. 4B:Recombinant caspase-3 was incubated with Ac-DEVD-AFC substrate in thepresence or absence of GST-XIAP, GST-BmIAP or 0.5 uM GST-SfIAP. FIG. 4C:Recombinant caspase-7 was incubated with Ac-DEVD-AFC substrate in thepresence or absence of GST-XIAP, GST-BmIAP or GST-SfIAP. Full details inExample 1, below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The invention provides polypeptides that are “inhibitors of apoptosisprotein (IAP) family members,” including the exemplary BmIAP (SEQ IDNO:2), and nucleic acids encoding them (e.g., SEQ ID NO:1), and methodsfor making and using these compositions. BmIAP was initially derivedfrom lepidopteran Bombyx mori BmN cells.

The polypeptides and nucleic acids of the invention can be used forinhibiting caspases, including insect, plant and mammalian caspases,such as human caspase 9, and “programmed cell death,” or apoptosis. Thepolypeptides of the invention have some amino acid sequence similaritywith SfIAP (see, e.g., Huang (2000) Proc. Natl. Acad. Sci. USA 2597:1427-1432), TnIAP and baculoviral CpIAP. As discussed herein,structure-function analysis of BmIAP (SEQ ID NO:2) reveals identicaldomain requirements (e.g. BIR plus RING, see FIG. 1A) for suppression ofapoptosis in both insect and mammalian cells, and similar caspaseselectivity (e.g. caspase-9) compared to the previously characterizedlepidopteran and baculovirus IAPs. These results strongly support theidea that lepidopteran IAPs are evolutionary conserved in both sequenceand function. Accordingly, the compositions and methods of the inventionare used to manipulate apoptotic (“cell death”) mechanisms in a varietyof cell types, including insect, plant and mammalian, such as human,cells, and organisms.

Drosophila and mammalian IAPs have been shown to play important roles inthe development of these organisms (see, e.g., Hay (1995) Cell83:1253-1262; Holcik (2000) Proc. Natl. Acad. Sci. USA 97(5),2286-2290). By analogy, the exemplary BmIAP (SEQ ID NO:2) is also acritical player in silkworm development. Accordingly, the compositionsand methods of the invention can be used to manipulate apoptosis duringthe development of the silkworm. Lepidopteran and coleopteran cells,e.g., Bombyx mori cells and Spodoptera frugiperda cells, are commonlyused in conjunction with expression vectors (recombinant viruses) toexpress large quantities of exogenous polypeptides (see, e.g., Juntunen(1999) Biochem J. 344 Pt 2:297-303); the compositions and methods of theinvention can be used to manipulate these expression systems. Thus, thecompositions and methods of the invention will have a significant impacton sericulture and biotechnology industries, particularly those relatedto the silkworm.

Example 1, below, describes the isolation and characterization of anovel group of “Inhibitor of Apoptosis Proteins (IAP),” designatedBmIAP. An exemplary polypeptide of this BmIAP group has a sequence asset forth in SEQ ID NO:2; an exemplary nucleic acid encodes it having asequence as set forth in SEQ ID NO:1. The exemplary IAP polypeptide andnucleic acid of the invention were initially derived from Bombyx moriBmN cells. BmIAP (SEQ ID NO:2) contains two baculoviral IAP repeat (BIR)domains followed by a RING domain (the BIR domain contains a highlyconserved arrangement of Cys/His residues forming a stable fold thatchelates zinc and the RING finger is a common zinc-binding motif thatalso exists in other cellular proteins), see FIG. 1A. BmIAP shares somecommon structural and functional properties with lepidopteran IAPs,SfIAP and TnIAP, and with two baculoviral IAPs, CpIAP and CpIAP,suggesting evolutionary conservation.

The polypeptides of the invention, “BmIAP,” block programmed cell death(apoptosis) in Spodoptera frugiperda Sf-21 cells induced by p35(apoptosis inhibiting)-deficient Autographa californicanucleopolyhedrovirus (AcMNPV). BmIAP's anti-apoptotic function requiresboth the BIR domains and RING domain. In mammalian cells, BmIAP inhibitsBax-induced but not Fas-induced apoptosis. The data discussed belowdemonstrates that BmIAP can inhibit mammalian caspase-9 (an initiatorcaspase in the mitochondria/cytochrome-c pathway), and may be a specificinhibitor of caspase 9, but not the downstream effector proteases,caspase-3 and caspase-7. While the invention is not limited by anyparticular mechanism of action, these data support the role of thepolypeptides of the invention in suppressing apoptosis as involvinginhibition of an upstream initiator caspase (e.g., mammalian caspase-9)in the conserved mitochondria/cytochrome-c pathway. Inhibition of suchcaspases effectively also inhibit apoptosis.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. As used herein, the following terms havethe meanings ascribed to them unless specified otherwise.

The term “antibody” or “Ab” includes both intact antibodies having atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds and antigen binding fragments thereof, or equivalentsthereof, either isolated from natural sources, recombinantly generatedor partially or entirely synthetic. Examples of antigen bindingfragments include, e.g., Fab fragments, F(ab′)2 fragments, Fd fragments,dAb fragments, isolated complementarity determining regions (CDR),single chain antibodies, chimeric antibodies, humanized antibodies,human antibodies made in non-human animals (e.g., transgenic mice) orany form of antigen binding fragment.

The terms “array” or “microarray” or “DNA array” or “nucleic acid array”or “biochip” as used herein is a plurality of target elements, eachtarget element comprising a defined amount of one or more nucleic acidmolecules, including the nucleic acids of the invention, immobilized asolid surface for hybridization to sample nucleic acids, as described indetail, below. The nucleic acids of the invention can be incorporatedinto any form of microarray, as described, e.g., in U.S. Pat. Nos.6,045,996; 6,022,963; 6,013,440; 5,959,098; 5,856,174; 5,770,456;5,556,752; 5,143,854.

A “biotic insult”, as used herein, refers to plant challenge caused byviable or biologic agents (biotic agents), such as insects, fungi,bacteria, viruses, nematodes, viroids, mycoplasmas, and the like.

An “abiotic insult”, as used herein, refers to plant challenge by anon-viable or non-living agent (abiotic agent). Abiotic agents that cancause an abiotic insult include, for example, environmental factors suchas low moisture (drought), high moisture (flooding), nutrientdeficiency, radiation levels, air pollution (ozone, acid rain, sulfurdioxide, etc.), temperature (hot and cold extremes), and soil toxicity,as well as herbicide damage, pesticide damage, or other agriculturalpractices (e.g., over-fertilization, improper use of chemical sprays,etc.).

The term “expression cassette” refers to any recombinant expressionsystem for the purpose of expressing a nucleic acid sequence of theinvention in vitro or in vivo, constitutively or inducibly, in any cell,including, in addition to insect and plant cells, prokaryotic, yeast,fungal or mammalian cells. The term includes linear or circularexpression systems. The term includes all vectors. The cassettes canremain episomal or integrate into the host cell genome. The expressioncassettes can have the ability to self-replicate or not, i.e., driveonly transient expression in a cell. The term includes recombinantexpression cassettes that contain only the minimum elements needed fortranscription of the recombinant nucleic acid.

The term “heterologous” when used with reference to a nucleic acid,indicates that the nucleic acid is in a cell or plant where it is notnormally found in nature; or, comprises two or more subsequences whichare not found in the same relationship to each other as normally foundin nature, or is recombinantly engineered so that its level ofexpression, or physical relationship to other nucleic acids or othermolecules in a cell, or structure, is not normally found in nature. Forinstance, a heterologous nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged ina manner not found in nature; e.g., a promoter sequence operably linkedto a nucleic of the invention. As another example, the inventionprovides recombinant constructs (expression cassettes, vectors, viruses,and the like) comprising various combinations of promoters and sequencesof the invention.

As used herein, “isolated,” when referring to a molecule or composition,such as, e.g., a nucleic acid or polypeptide of the invention, meansthat the molecule or composition is separated from at least one othercompound, such as a protein, DNA, RNA, or other contaminants with whichit is associated in vivo or in its naturally occurring state. Thus, anucleic acid sequence is considered isolated when it has been isolatedfrom any other component with which it is naturally associated. Anisolated composition can, however, also be substantially pure. Anisolated composition can be in a homogeneous state. It can be in a dryor an aqueous solution. Purity and homogeneity can be determined, e.g.,using analytical chemistry techniques such as, e.g., polyacrylamide gelelectrophoresis (SDS-PAGE) or high performance liquid chromatography(HPLC).

The term “nucleic acid” or “nucleic acid sequence” refers to adeoxy-ribonucleotide or ribonucleotide oligonucleotide, includingsingle- or double-stranded forms, and coding or non-coding (e.g.,“antisense”) forms. The term encompasses nucleic acids containing knownanalogues of natural nucleotides. The term also encompassesnucleic-acid-like structures with synthetic backbones. DNA backboneanalogues provided by the invention include phosphodiester,phosphorothioate, phosphorodithioate, methylphosphonate,phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal,methylene(methylimino), 3′-N-carbamate, morpholino carbamate, andpeptide nucleic acids (PNAs); see Oligonucleotides and Analogues, aPractical Approach, edited by F. Eckstein, IRL Press at OxfordUniversity Press (1991); Antisense Strategies, Annals of the New YorkAcademy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992);Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research andApplications (1993, CRC Press). PNAs contain non-ionic backbones, suchas N-(2-aminoethyl) glycine units. Phosphorothioate linkages aredescribed, e.g., by U.S. Patent Nos. 6,031,092; 6,001,982; 5,684,148;see also, WO 97/0321 1; WO 96/39154; Mata (1 997) Toxicol. Appl.Pharmacol. 144:189-197. Other synthetic backbones encompassed by theterm include methyl-phosphonate linkages or alternatingmethylphosphonate and phosphodiester linkages (see, e.g., U.S. Pat. No.5,962,674; Strauss-Soukup (1997) Biochemistry 36:8692-8698), andbenzylphosphonate linkages (see, e.g., U.S. Pat. No. 5,532,226; Samstag(1996) Antisense Nucleic Acid Drug Dev 6:153-156). The term nucleic acidis used interchangeably with gene, DNA, RNA, cDNA, mRNA, oligonucleotideprimer, probe and amplification product.

As used herein the terms “polypeptide,” “protein,” and “peptide” areused interchangeably and include compositions of the invention that alsoinclude “analogs,” or “conservative variants” and “mimetics” (e.g.,“peptidomimetics”) with structures and activity that substantiallycorrespond to the polypeptides of the invention, including the exemplarysequence as set forth in SEQ ID NO:2. Thus, the terms “conservativevariant” or “analog” or “mimetic” also refer to a polypeptide or peptidewhich has a modified amino acid sequence, such that the change(s) do notsubstantially alter the polypeptide's (the conservative variant's)structure and/or activity (e.g., ability to inhibit caspase 9, toinhibit apoptosis), as defined herein. These include conservativelymodified variations of an amino acid sequence, i.e., amino acidsubstitutions, additions or deletions of those residues that are notcritical for protein activity, or substitution of amino acids withresidues having similar properties (e.g., acidic, basic, positively ornegatively charged, polar or non-polar, etc.) such that thesubstitutions of even critical amino acids does not substantially alterstructure and/or activity. Conservative substitution tables providingfunctionally similar amino acids are well known in the art. For example,one exemplary guideline to select conservative substitutions includes(original residue followed by exemplary substitution): ala/gly or ser;arg/lys; asn/gln or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala orpro; his/asn or gln; ile/leu or val; leu/ile or val; lys/arg or gln orglu; met/leu or tyr or ile; phe/met or leu or tyr; ser/thr; thr/ser;trp/tyr; tyr/trp or phe; val/ile or leu. An alternative exemplaryguideline uses the following six groups, each containing amino acidsthat are conservative substitutions for one another: 1) Alanine (A),Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3)Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (see also, e.g.,Creighton (1984) Proteins, W. H. Freeman and Company; Schulz and Schimer(1979) Principles of Protein Structure, Springer-Verlag). One of skillin the art will appreciate that the above-identified substitutions arenot the only possible conservative substitutions. For example, for somepurposes, one may regard all charged amino acids as conservativesubstitutions for each other whether they are positive or negative. Inaddition, individual substitutions, deletions or additions that alter,add or delete a single amino acid or a small percentage of amino acidsin an encoded sequence can also be considered “conservatively modifiedvariations.”

The terms “mimetic” and “peptidomimetic” refer to a synthetic chemicalcompound that has substantially the same structural and/or functionalcharacteristics of the polypeptides of the invention (e.g., ability toinhibit apoptosis, antigenicity, etc.). The mimetic can be eitherentirely composed of synthetic, non-natural analogues of amino acids,or, is a chimeric molecule of partly natural peptide amino acids andpartly non-natural analogs of amino acids. The mimetic can alsoincorporate any amount of natural amino acid conservative substitutionsas long as such substitutions also do not substantially alter themimetics' structure and/or activity. As with polypeptides of theinvention which are conservative variants, routine experimentation willdetermine whether a mimetic is within the scope of the invention, i.e.,that its structure and/or function is not substantially altered.Polypeptide mimetic compositions can contain any combination ofnon-natural structural components, which are typically from threestructural groups: a) residue linkage groups other than the naturalamide bond (“peptide bond”) linkages; b) non-natural residues in placeof naturally occurring amino acid residues; or c) residues which inducesecondary structural mimicry, i.e., to induce or stabilize a secondarystructure, e.g., a beta turn, gamma turn, beta sheet, alpha helixconformation, and the like. A polypeptide can be characterized as amimetic when all or some of its residues are joined by chemical meansother than natural peptide bonds. Individual peptidomimetic residues canbe joined by peptide bonds, other chemical bonds or coupling means, suchas, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctionalmaleimides, N,N′-dicyclohexylcarbodiimide (DCC) orN,N′-diisopropylcarbodiimide (DIC). Linking groups that can be analternative to the traditional amide bond (“peptide bond”) linkagesinclude, e.g., ketomethylene (e.g., —C(═O)—CH₂— for —C(═O)—NH—),aminomethylene (CH₂—NH), ethylene, olefin (CH═CH), ether (CH₂—O),thioether (CH₂—S), tetrazole (CN4—), thiazole, retroamide, thioamide, orester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of AminoAcids, Peptides and Proteins, Vol. 7, pp 267-357, “Peptide BackboneModifications,” Marcell Dekker, N.Y.). A polypeptide can also becharacterized as a mimetic by containing all or some non-naturalresidues in place of naturally occurring amino acid residues;non-natural residues are well described in the scientific and patentliterature.

As used herein, a “pathogen” refers to any agent that causes a diseaseor disease state in an animal or plant, including, but not limited toviruses, fungi, bacterium, nematodes, and other related microorganisms.

The term “plant” includes whole plants, plant parts (e.g., leaves,stems, flowers, roots, etc.), plant protoplasts, seeds and plant cellsand progeny of same. The class of plants which can be used in the methodof the invention is generally as broad as the class of higher plantsamenable to transformation techniques, including angiosperms(monocotyledonous and dicotyledonous plants), as well as gymnosperms. Itincludes plants of a variety of ploidy levels, including polyploid,diploid, haploid and hemizygous states. Plantlets are also includedwithin the meaning of “plant”. Suitable plants for use in the inventioninclude any plants amenable to transformation techniques, including bothmonocotyledonous and dicotyledonous plants. Examples of monocotyledonousplants include, but are not limited to, asparagus, field and sweet corn,barley, wheat, rice, sorghum, onion, pearl millet, rye and oat, andornamentals. Examples of dicotyledonous plants include, but are notlimited to, tomato, potato, arabidopsis, tobacco, cotton, rapeseed,field beans, soybeans, peppers, lettuce, peas, alfalfa, clover, colecrops or Brassica (e.g., cabbage, broccoli, cauliflower, brusselsprouts), radish, carrot, beets, eggplant, spinach, cucumber, squash,melons, cantaloupe, sunflowers and various ornamentals. The term “plantcell”, as used herein, refers to protoplasts, gamete producing cells,and cells that are capable of regenerating into whole plants.Accordingly, a seed comprising multiple plant cells capable ofregenerating into a whole plant is included in the definition of “plantcell”. As used herein, “plant tissue” includes differentiated andundifferentiated tissues of a plant, including but not limited to roots,stems, shoots, leaves, pollen, seeds, tumor tissue and various forms ofcells and culture such as single cells, protoplast, embryos, and callustissue.

As used herein, “recombinant” refers to a polynucleotide synthesized orotherwise manipulated in vitro (e.g., “recombinant polynucleotide”), tomethods of using recombinant polynucleotides to produce gene products incells or other biological systems, or to a polypeptide (“recombinantprotein”) encoded by a recombinant polynucleotide.

As used herein, the term “promoter” includes all sequences capable ofdriving transcription of a coding sequence in a cell, including aninsect cell, a plant cell, a mammalian cell, and the like. Thus,promoters used in the constructs of the invention include cis-actingtranscriptional control elements and regulatory sequences that areinvolved in regulating or modulating the timing and/or rate oftranscription of a gene. For example, a promoter can be a cis-actingtranscriptional control element, including an enhancer, a promoter, atranscription terminator, an origin of replication, a chromosomalintegration sequence, 5′ and 3′ untranslated regions, or an intronicsequence, which are involved in transcriptional regulation. Thesecis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.)transcription.

The term percent “sequence identity,” in the context of two or morenucleic acids or polypeptide sequences refers to two or more sequencesor subsequences that are the same or have a specified percentage ofnucleotides (or amino acid residues) that are the same, when comparedand aligned for maximum correspondence over a comparison window, asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. This definition also refers tothe complement (antisense strand) of a sequence. For example, inalternative embodiments, nucleic acids within the scope of the inventioninclude those with a nucleotide sequence identity that is at least about95%, at least about 96%, at least about 97%, at least about 98%, atleast about 99% of the exemplary sequence set forth in SEQ ID NO:1. Inalternative embodiments, polypeptides within the scope of the inventioninclude those with an amino acid sequence identity that is at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,at least about 99% of the exemplary sequences set forth in SEQ ID NO:2.Two sequences with these levels of identity are “substantiallyidentical” and within the scope of the invention. Thus, if a nucleicacid sequence has the requisite sequence identity to SEQ ID NO:1, or asubsequence thereof, it also is a polynucleotide sequence within thescope of the invention. If a polynucleotide sequence has the requisitesequence identity to SEQ ID NO:2, or a subsequence thereof, it also is apolypeptide within the scope of the invention. In one aspect, thepercent identity exists over a region of the sequence that is at leastabout 25 nucleotides or amino acid residues in length, or, over a regionthat is at least about 50 to 100 nucleotides or amino acids in length.Parameters (including, e.g., window sizes, gap penalties and the like)to be used in calculating “percent sequence identities” between twonucleic acids or polypeptides to identify and determine whether one iswithin the scope of the invention are described in detail, below.

Polypeptides and Peptides

The invention provides an isolated or recombinant polypeptide comprisinga sequence having at least 95% sequence identity to SEQ ID NO:2. Oneexemplary polypeptide comprises the sequence as set forth in SEQ IDNO:2, and fragments (e.g., antigenic fragments) thereof (as noted above,the term polypeptide includes peptides and peptidomimetics, etc.).Polypeptides and peptides of the invention can be isolated from naturalsources, be synthetic, or be recombinantly generated polypeptides.Peptides and proteins can be recombinantly expressed in vitro or invivo. The peptides and polypeptides of the invention can be made andisolated using any method known in the art.

Polypeptide and peptides of the invention can also be synthesized, wholeor in part, using chemical methods well known in the art. See e.g.,Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980)Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K., TherapeuticPeptides and Proteins, Formulation, Processing and Delivery Systems(1995) Technomic Publishing Co., Lancaster, Pa. For example, peptidesynthesis can be performed using various solid-phase techniques (seee.g., Roberge (1995) Science 269:202; Merrifield (1997) Methods Enzymol.289:3-13) and automated synthesis may be achieved, e.g., using the ABI431A Peptide Synthesizer (Perkin Elmer). The skilled artisan willrecognize that individual synthetic residues and polypeptidesincorporating mimetics can be synthesized using a variety of proceduresand methodologies, which are well described in the scientific and patentliterature, e.g., Organic Syntheses Collective Volumes, Gilman, et al.(Eds) John Wiley & Sons, Inc., NY. Polypeptides incorporating mimeticscan also be made using solid phase synthetic procedures, as described,e.g., by Di Marchi, et al., U.S. Pat. No. 5,422,426. Peptides andpeptide mimetics of the invention can also be synthesized usingcombinatorial methodologies. Various techniques for generation ofpeptide and peptidomimetic libraries are well known, and include, e.g.,multipin, tea bag, and split-couple-mix techniques; see, e.g., al-Obeidi(1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol.1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996)Methods Enzymol. 267:220-234. Modified peptides of the invention can befurther produced by chemical modification methods, see, e.g., Belousov(1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol.Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896.

The invention provides a fusion protein comprising a polypeptide of theinvention, e.g., a sequence having at least 95% sequence identity to SEQID NO:2, and a second domain. Thus, peptides and polypeptides of theinvention are synthesized and expressed as chimeric or “fusion” proteinswith one or more additional domains linked thereto for, e.g, to morereadily isolate or identify a recombinantly synthesized peptide, and thelike. Detection and purification facilitating domains include, e.g,metal chelating peptides such as polyhistidine tracts andhistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wass.). The inclusion of acleavable linker sequences such as Factor Xa or enterokinase(Invitrogen, San Diego Calif.) between the purification domain andGCA-associated peptide or polypeptide can be useful to facilitatepurification. For example, an expression vector can include anepitope-encoding nucleic acid sequence linked to six histidine residuesfollowed by a thioredoxin and an enterokinase cleavage site (see, e.g.,Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr.Purif. 12:404-14). The histidine residues facilitate detection andpurification while the enterokinase cleavage site provides a means forpurifying the epitope from the remainder of the fusion protein.

Nucleic Acids, Expression Vectors and Transformed Cells

The invention provides an isolated or recombinant nucleic acidcomprising a nucleic acid sequence having at least 95% sequence identityto SEQ ID NO:1, and expression cassettes (e.g., vectors), cells andtransgenic animals comprising the nucleic acids of the invention. As thegenes and vectors of the invention can be made and expressed in vitro orin vivo, the invention provides for a variety of means of making andexpressing these genes and vectors. One of skill will recognize thatdesired phenotypes associated with altered gene activity can be obtainedby modulating the expression or activity of the genes and nucleic acids(e.g., promoters) within the expression cassettes (e.g., vectors) of theinvention. Any of the known methods described for increasing ordecreasing expression or activity can be used for this invention. Theinvention can be practiced in conjunction with any method or protocolknown in the art, which are well described in the scientific and patentliterature.

The nucleic acid sequences of the invention and other nucleic acids usedto practice this invention, whether RNA, cDNA, genomic DNA, vectors,viruses or hybrids thereof, may be isolated from a variety of sources,genetically engineered, amplified, and/or expressed recombinantly. Anyrecombinant expression system can be used, including, in addition toinsect and bacterial cells, e.g., mammalian, yeast or plant cellexpression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g.,Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) FreeRadic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896;Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109;Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,generating mutations in sequences, subcloning, labeling probes,sequencing, hybridization and the like are well described in thescientific and patent literature, see, e.g., Sambrook, ed., MOLECULARCLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring HarborLaboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed.John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES INBIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACIDPROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed.Elsevier, N.Y. (1993).

The invention provides nucleic acids of the invention “operably linked”to a transcriptional regulatory sequence. “Operably linked” refers to afunctional relationship between two or more nucleic acid (e.g., DNA)segments. Typically, it refers to the functional relationship of atranscriptional regulatory sequence to a transcribed sequence. Forexample, a promoter is operably linked to a coding sequence, such as anucleic acid of the invention, if it stimulates or modulates thetranscription of the coding sequence in an appropriate host cell orother expression system. Generally, promoter transcriptional regulatorysequences that are operably linked to a transcribed sequence arephysically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory sequences, such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance. Forexample, in one embodiment, a promoter is operably linked to a nucleicacid sequence of the invention, as exemplified by SEQ ID NO:1.

The invention further provides cis-acting transcriptional regulatorysequences, which, in vivo, are operably linked to the coding sequencefor the exemplary polypeptide of the invention, SEQ ID NO:2, includingpromoters, comprising the genomic sequences 5′ (upstream) of atranscriptional start site (see SEQ ID NO:1) and intronic sequences. Thepromoters of the invention contain cis-acting transcriptional regulatoryelements involved in message expression. These promoter sequences may bereadily obtained using routine molecular biological techniques. Forexample, additional genomic (and promoter) sequences may be obtained byscreening Bombyx mori genomic libraries using nucleic acids of theinvention. For example, genomic sequence can be readily identified by“chromosome walking” techniques, as described by, e.g., Hauser (1998)Plant J 16:117-125; Min (1998) Biotechniques 24:398-400. Other usefulmethods for further characterization of promoter sequences include thosegeneral methods described by, e.g., Pang (1997) Biotechniques22:1046-1048; Gobinda (1993) PCR Meth. Applic. 2:318; Triglia (1988)Nucleic Acids Res. 16:8186; Lagerstrom (1991) PCR Methods Applic. 1:111;Parker (1991) Nucleic Acids Res. 19:3055. As is apparent to one ofordinary skill in the art, these techniques can also be applied toidentify, characterize and isolate any genomic or cis-acting regulatorysequences corresponding to or associated with the nucleic acid andpolypeptide sequences of the invention.

The invention provides oligonucleotide primers that can amplify all orany specific region within a nucleic acid sequence of the invention,particularly, the exemplary SEQ ID NO:1. The nucleic acids of theinvention can also be mutated, detected, generated or measuredquantitatively using amplification techniques. Using the nucleic acidsequences of the invention (e.g., as in the exemplary SEQ ID NO:1), theskilled artisan can select and design suitable oligonucleotideamplification primers. Amplification methods are also known in the art,and include, e.g., polymerase chain reaction, PCR (see, e.g., PCRPROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, AcademicPress, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press,Inc., N.Y.); ligase chain reaction (LCR) (see, e.g., Barringer (1990)Gene 89:117); transcription amplification (see, e.g., Kwoh (1989) Proc.Natl. Acad. Sci. USA, 86:1173); and, self-sustained sequence replication(see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA, 87:1874); Q Betareplicase amplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491; Burg (1996) Mol. Cell. Probes 10:257-271) and other RNApolymerase mediated techniques (e.g., NASBA, Cangene, Mississauga,Ontario).

Expression vectors capable of expressing the nucleic acids andpolypeptides of the invention in animal cells, including insect andmammalian cells, are well known in the art. Vectors which may beemployed include recombinantly modified enveloped or non-enveloped DNAand RNA viruses, e.g., from baculoviridiae, parvoviridiae,picornoviridiae, herpesveridiae, poxviridae, adenoviridiae,picornnaviridiae or alphaviridae. Insect cell expression systemscommonly use recombinant variations of baculoviruses and othernucleopolyhedrovirus, e.g., Bombyx mori nucleopolyhedrovirus vectors(see, e.g., Choi (2000) Arch. Virol. 145:171-177). For example,Lepidopteran and Coleopteran cells are used to replicate baculovirusesto promote expression of foreign genes carried by baculoviruses, e.g.,Spodoptera frugiperda cells are infected with recombinant Autographacalifornica nuclear polyhedrosis viruses (AcNPV) carrying aheterologous, e.g., a human, coding sequence (see, e.g., Lee (2000) J.Virol. 74:11873-11880; Wu (2000) J. Biotechnol. 80:75-83). See, e.g.,U.S. Pat. No. 6,143,565, describing use of the polydnavirus of theparasitic wasp Glyptapanteles indiensis to stably integrate nucleic acidinto the genome of Lepidopteran and Coleopteran insect cell lines. Seealso, U.S. Pat. Nos. 6,130,074; 5,858,353; 5,004,687.

Mammalian expression vectors can be derived from adenoviral,adeno-associated viral or retroviral genomes. Retroviral vectors caninclude those based upon murine leukemia virus (see, e.g., U.S. Pat. No.6,132,731), gibbon ape leukemia virus (see, e.g., U.S. Pat. No.6,033,905), simian immuno-deficiency virus, human immuno-deficiencyvirus (see, e.g., U.S. Pat. No. 5,985,641), and combinations thereof.Describing adenovirus vectors, see, e.g., U.S. Pat. Nos. 6,140,087;6,136,594; 6,133,028; 6,120,764. See, e.g., Okada (1996) Gene Ther.3:957-964; Muzyczka (1994) J. Clin. Invst. 94:1351; U.S. Pat. Nos.6,156,303; 6,143,548 5,952,221, describing AAV vectors. See also6,004,799; 5,833,993.

Expression vectors capable of expressing proteins in plants are wellknown in the art, and can include, e.g., vectors from Agrobacteriumspp., potato virus X (see, e.g., Angell (1997) EMBO J. 16:3675-3684),tobacco mosaic virus (see, e.g., Casper (1996) Gene 173:69-73), tomatobushy stunt virus (see, e.g, Hillman (1989) Virology 169:42-50), tobaccoetch virus (see, e.g., Dolja (1997) Virology 234:243-252), bean goldenmosaic virus (see, e.g., Morinaga (1993) Microbiol Immunol. 37:471-476),cauliflower mosaic virus (see, e.g., Cecchini (1997) Mol. Plant MicrobeInteract. 10:1094-1101), maize Ac/Ds transposable element (see, e.g.,Rubin (1997) Mol. Cell. Biol. 17:6294-6302; Kunze (1996) Curr. Top.Microbiol. Immunol. 204:161-194), and the maize suppressor-mutator (Spm)transposable element (see, e.g., Schlappi (1996) Plant Mol. Biol.32:717-725); and derivatives thereof.

The invention provides a transformed cell comprising a nucleic acid ofthe invention. The cells can be mammalian (such as human), insect (suchas Spodoptera frugiperda, Spodoptera exigua, Spodoptera littoralis,Spodoptera litura, Pseudaletia separata, Trichoplusia ni, Plutellaxylostella, Bombyx mori, Lymantria dispar, Heliothis virescens,Autographica californica and other insect, particularly lepidopteran andcoleopteran, cell lines), plant, bacterial, yeast, and the like.Techniques for transforming and culturing cells are well described inthe scientific and patent literature; see, e.g., Weiss (1995) MethodsMol. Biol. 39:79-95, describing insect cell culture in serum-free media;Tom (1995) Methods Mol. Biol. 39:203-224; Kulakosky (1998) Glycobiology8:741-745; Altmann (1999) Glycoconj. J. 16:109-123; Yanase (1998) ActaVirol. 42:293-298; U.S. Pat. Nos. 6,153,409; 6,143,565; 6,103,526.

Transgenic Non-human Animals

The invention also provides transgenic animals, including mammals andinsects. Insects stably expressing the nucleic acids of the inventioncan be used for, e.g., experiments studies on apoptosis, screening formodulators of caspases and apoptosis, manipulation of insect lifecycles, such as Bombyx mori and its use in silk production. The nucleicacids of the invention can be expressed in a variety of insect larvae,e.g., Bombyx mori (see, e.g., Maeda (1985) Nature 315: 592-594),Trichoplusia ni, the cabbage looper larvae (Medin (1990) Proc. Nat.Acad. Sci. USA 87: 2760-2764) and Manduca sexta, the tobacco hornworm(U.S. Pat. No. 5,471,858). See, e.g., Keshan (2000) J. Insect PhysioL46:1061-1068; U.S. Pat. No. 5,118,616.

The invention also provides transgenic non-human mammals, e.g., goats,rats and mice, comprising the chimeric nucleic acids of the invention.These animals can be used, e.g., as in vivo models to study apoptosis,or, as models to screen for modulators of caspase enzyme activity invivo. Transgenic non-human animals can be designed and generated usingany method known in the art; see, e.g., U.S. Pat. Nos. 6,156,952;6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854; 5,892,070;5,880,327; 5,891,698; 5,639,940; 5,573,933, describing making and usingtransgenic mice, rats, rabbits, sheep, pigs and cows. See also, e.g.,Pollock (1999) J. Immunol. Methods 231:147-157, describing theproduction of recombinant proteins in the milk of transgenic dairyanimals; Baguisi (1999) Nat. Biotechnol. 17:456-461, demonstrating theproduction of transgenic goats.

Transgenic Plants

The invention also provides transgenic plants, including seeds,expressing the nucleic acids and polypeptide of the invention. Nucleicacids may be introduced into the genome of the desired plant host by avariety of conventional techniques. For example, the DNA construct maybe introduced directly into the genomic DNA of the plant cell usingtechniques such as electroporation and microinjection of plant cellprotoplasts, or the DNA constructs can be introduced directly to planttissue using ballistic methods, such as DNA particle bombardment.

Alternatively, transformed plant cells can be generated by fusion of therecipient cells with bacterial protoplasts containing DNA, use of DEAEdextran, polyethylene glycol precipitation, as described, e.g., inPaszkowski (1984) EMBO J. 3:2717-2722. DNA construct can be introduceddirectly into the genomic DNA of the plant cell using electroporation,as described, e.g., in Fromm (1985) Proc. Natl. Acad. Sci. USA 82:5824,or by microinjection of plant cell protoplasts, as described, e.g.,Schnorf (1991) Transgenic Res. 1:23-30.

Nucleic acids can be introduced directly to plant tissue using ballisticmethods, such as DNA particle bombardment. Microprojectile bombardmentto deliver DNA into plant cells is an alternative means oftransformation for the numerous species considered recalcitrant toAgrobacterium- or protoplast-mediated transformation methods. Forexample, see, e.g., Christou (1997) Plant Mol. Biol. 35:197-203;Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987) Nature327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discussing use ofparticle bombardment to introduce transgenes into wheat; and Adam (1997)supra, for use of particle bombardment to introduce YACs into plantcells. For example, Rinehart (1997) supra, used particle bombardment togenerate transgenic cotton plants. Apparatus for accelerating particlesis described U.S. Pat. No. 5,015,580; and, the commercially availableBioRad (Biolistics) PDS-2000 particle acceleration instrument; see also,John, U.S. Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5, 681,730,describing particle-mediated transformation of gymnosperms.

DNA can also be introduced in to plant cells using recombinant viruses.Plant cells can be transformed using viral vectors, such as, e.g.,tobacco mosaic virus derived vectors (Rouwendal (1997) Plant Mol. Biol.33:989-999), see Porta (1996) “Use of viral replicons for the expressionof genes in plants,” Mol. Biotechnol. 5:209-221.

Alternatively, the DNA constructs may be combined with suitable T-DNAflanking regions and introduced into a conventional Agrobacteriumtumefaciens host vector. The virulence functions of the Agrobacteriumtumefaciens host will direct the insertion of the construct and adjacentmarker into the plant cell DNA when the cell is infected by thebacteria. Agrobacterium tumefaciens-mediated transformation techniques,including disarming and use of binary vectors, are well described in thescientific literature. See, e.g., Horsch (1984) Science 233:496-498;Fraley (1983) Proc. Natl. Acad. Sci. USA 80:4803 (1983); Gene Transferto Plants, Potrykus, ed. (Springer-Verlag, Berlin 1995).

Alignment Analysis of Sequences

The nucleic acid sequences of the invention include genes and geneproducts identified and characterized by analysis using the exemplarynucleic acid and protein sequences of the invention, including SEQ IDNO:1 and SEQ ID NO:2. For sequence comparison, typically one sequenceacts as a reference sequence, to which test sequences are compared. Whenusing a sequence comparison algorithm, test and reference sequences areentered into a computer, subsequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated.Default program parameters are used unless alternative parameters aredesignated herein. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated or default programparameters. A “comparison window”, as used herein, includes reference toa segment of any one of the number of contiguous positions selected fromthe group consisting of from 25 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2:482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), bycomputerized implementations of these algorithms (CLUSTAL, GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Dr., Madison, Wis.), or by manual alignmentand visual inspection.

In one aspect, a CLUSTAL algorithm, such as the CLUSTAL W program, isused to determine if a nucleic acid or polypeptide sequence is withinthe scope of the invention; see, e.g., Thompson (1994) Nuc. Acids Res.22:4673-4680; Higgins (1996) Methods Enzymol 266:383-402. Variations canalso be used, such as CLUSTAL X, see Jeanmougin (1998) Trends BiochemSci 23:403-405; Thompson (1997) Nucleic Acids Res 25:4876-4882. CLUSTALW program, described by Thompson (1994) supra, in the methods of theinvention used with the following parameters: K tuple (word) size: 1,window size: 5, scoring method: percentage, number of top diagonals: 5,gap penalty: 3.

Another algorithm is PILEUP, which can be used to determine whether apolypeptide or nucleic acid has sufficient sequence identity to SEQ IDNO:1 or SEQ ID NO:2 to be with the scope of the invention. This programcreates a multiple sequence alignment from a group of related sequencesusing progressive, pairwise alignments to show relationship and percentsequence identity. It also plots a tree or dendogram showing theclustering relationships used to create the alignment. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360 (1987). The method used is similar to themethod described by Higgins & Sharp, CABIOS 5:151-153 (1989). Thefollowing parameters are used with PILEUP in the methods of theinvention: default gap weight (3.00), default gap length weight (0.10),and weighted end gaps.

Another example of an algorithm that is suitable for determining percentsequence identity (i.e., substantial similarity or identity) in thisinvention is the BLAST algorithm, which is described in Altschul (1990)J. Mol. Biol. 215:403-410. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul (1990) supra). These initial neighborhood wordhits act as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues, always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. In oneembodiment, to determine if a nucleic acid sequence is within the scopeof the invention, the BLASTN program (for nucleotide sequences) is usedincorporating as defaults a wordlength (W) of 11, an expectation (E) of10, M=5, N=4, and a comparison of both strands. For amino acidsequences, the BLASTP program uses as default parameters a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix(see, e.g., Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

Antibodies

The invention provides antibodies that specifically bind to thepolypeptides of the invention, e.g., the exemplary SEQ ID NO:2. Theseantibodies can be used, e.g., to isolate the polypeptides of theinvention, to identify the presence of polypeptides that are associatedwith apoptosis, and the like. To generate antibodies, polypeptides orpeptides (antigenic fragments of SEQ ID NO:2) can be conjugated toanother molecule or can be administered with an adjuvant. The codingsequence can be part of an expression cassette or vector capable ofexpressing the immunogen in vivo (see, e.g., Katsumi (1994) Hum. GeneTher. 5:1335-9). Methods of producing polyclonal and monoclonalantibodies are known to those of skill in the art and described in thescientific and patent literature, see, e.g., Coligan, CURRENT PROTOCOLSIN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASIC AND CLINICALIMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos, Calif.;Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) AcademicPress, New York, N.Y. (1986); Harlow (1988) ANTIBODIES, A LABORATORYMANUAL, Cold Spring Harbor Publications, New York.

Antibodies also can be generated in vitro, e.g., using recombinantantibody binding site expressing phage display libraries, in addition tothe traditional in vivo methods using animals. See, e.g. Huse (1989)Science 246:1275; Ward (1989) Nature 341:544; Hoogenboom (1997) TrendsBiotechnol. 15:62-70; Katz (1997) Annu. Rev. Biophys. Biomol. Struct.26:27-45. Human antibodies can be generated in mice engineered toproduce only human antibodies, as described by, e.g., U.S. Pat. No.5,877,397; 5,874,299; 5,789,650; and 5,939,598. B-cells from these micecan be immortalized using standard techniques (e.g., by fusing with animmortalizing cell line such as a myeloma or by manipulating suchB-cells by other techniques to perpetuate a cell line) to produce amonoclonal human antibody-producing cell. See, e.g., U.S. Pat. No.5,916,771; 5,985,615.

Measuring Markers of Apoptosis in a Cell

The invention provides a method for identifying an agent that canmodulate the activity of a polypeptide of the invention (e.g., apolypeptide having a sequence at least 95% sequence identity to SEQ IDNO:2) that is capable inhibiting apoptosis in a cell. The methodcomprises contacting a cell expressing the polypeptide recombinantly inthe presence and absence of a test agent before, during or afterinducing apoptosis in the cell; and, measuring the amount or degree ofthe polypeptide's activity in the cell in the presence and absence ofthe test agent, wherein an increase or decrease in the amount or degreeof apoptosis in the cell in the presence of the test agent identifiesthe test agent as a modulator of the polypeptide's activity.

The degree of the polypeptide's activity in the cell can be determinedby measuring the amount or degree of apoptosis in the cell; the amountor degree of caspase protease activity in the cell; the amount or degreeof DNA fragmentation in the cell; the amount or degree of cleavage ofsubstrates of caspases in the cell; or by measuring the amount or degreeof any surrogate marker of apoptosis in the cell; methods for makingthese measurements are well known in the art, and the inventionincorporates all such methods and variations thereof. For example, seeMethods in Enzymology (2000) volume 322, edited by John C. Reed,Academic Press, e.g., chapters on pages 3, 15, 41, describing assays tomeasure apoptosis and surrogate markers of apoptosis and enzymes withactivity related to levels of apoptosis, e.g., assays to determine DNAfragmentation, caspase assays, measuring annexin V (see, e.g., Zhang(1997) Biotechniques 23:525-531), and the like. See, e.g., van Engeland(1996) Cytometry 24:131-139; Gorczyca (1998) Methods Mol. Biol.91:217-238. See also, e.g., U.S. Pat. Nos. 6,165,737; 6,165,732;6,160,095; 6,143,522; 6,087,384; 6,077,684; 6,060,238; 6,054,436;5,985,829; 5,976,786; 5,952,189.

EXAMPLES

The following example is offered to illustrate, but not to limit theclaimed invention.

Example 1 Cloning, Recombinant Expression and Characterization of BmIAP

The following example describes the initial cloning of the BmIAP of theinvention, recombinant expression of BmIAP nucleic acids andpolypeptides and characterization of the BmIAP of the invention.

Cloning of BmIAP: mRNA was isolated from BmN cells by using a kit fromQiagen, Inc. (Valencia, Calif.). Degenerate primers were used; they weredesigned according to the consensus amino acid sequences betweenbaculoviral IAPs and Drosophila IAPs, having the sequence5′-GC(A/C/G/T)GA(A/C/G/T)GC(A/C/G/T)GG(A/C/G/T) TT(T/C)TT(T/C)TA-3′ (SEQID NO:3) and 5′-AC(A/C/G/T)AC(A/G)TG(A/C/G/T)CC (A/G)CA(A/C/G/T)GG-3′(SEQ ID NO:4). Reverse transcription-PCR was performed for 35 cycles byusing 94° C. for 45 sec, 46° C. for 1 min and 72° C. for 1 min.Amplified fragments were blunt-end-cloned into HincII site of pTZ-19 andthen sequenced. To obtain full-length BmIAP cDNAs, 5′ RACE and 3′ RACEwere performed by using commercial kits (GIBCO/BRL; Takara) and5′-CTGTTCCCACGGAACGTC-3′ (SEQ ID NO:5) and 5′-GCCACCAATGATTCGAC-3′ (SEQID NO:6) as internal PCR primers.

Plasmid construction: A cDNA fragment encompassing the complete ORF ofBmIAP was PCR-amplified and subcloned into the EcoRI-XhoI sites inpcDNA3-myc and pGEX4T-1. Plasmids encoding fragments of BmIAP, includingBIR1+2 (amino acid residue 1-292) (SEQ ID NO:2) and RING (residue293-346) (SEQ ID NO:2), were amplified by PCR by using primerscontaining either start or stop codons as appropriate and subcloned intopTZ-19 and pcDNA3-myc plasmids.

Protein expression and purification: pGEX4T-1-BmIAP plasmid wasintroduced into E. coli strain BL21 (DE3) containing the plasmid pT-Trx.Glutathione S-transferase (GST) fusion proteins were obtained byinduction with 0.05 mM isopropyl β-thiogalactoside at 25° C. for 8 hrand then purified using glutathione-Sepharose (see, e.g., Huang (2000)Proc. Natl. Acad. Sci. USA 97:1427-1432). The catalytic domains ofcaspase-3, caspase-7 and caspase-9 were expressed, purified byNi-chelation affinity-chromatography, and quantified as described byStennicke (1997) J. Biol. Chem. 272:25719-25723; Stennicke (1998) J.Biol. Chem 273:27084-27090; Stennicke (19999) J. Biol. Chem.274:8359-8362.

Cell extracts and Caspase assays: Cytosolic extracts were prepared byusing human embryonic kidney (HEK) 293 cells (see, e.g., Deveraux (1997)Nature 388:300-303). For initiating caspase activation, 1 uM horse heartcytochrome-c (Sigma) and 1 mM dATP was added to extracts, as describedby Deveraux (1998) Embo J. 17:2215-2223). Caspase activity was assayedby release of 7-amino4-trifluoromethyl-coumarin (AFC) from Ac-DEVD-AFCor Ac-LEHD-AFC (Calbiochem), using a spectrofluorimeter as described byStennicke (1997) supra; Quan (1995) J. Biol. Chem. 270:10377-10379.

Cell culture, Transfection and Apoptosis Assays: Insect Sf-21 cells weremaintained at 27° C. in Excell 401 medium (JRH Biosciences, Lenexa, KS)supplemented with 2.5% FBS. vP35del, Autographa californica nuclearpolyhedrosis virus (AcMNPV) containing a deletion in the apoptosissuppressor “p35” gene (35-kilodalton protein gene) (see, e.g., Lerch(1993) Nucleic Acids Res. 21:1753-1760) was propagated in TN-386 cells(see, e.g., Clem (1991) Science 254:1388-1390). Plasmids encodingfull-length or deletion mutants of BmIAP (1 ug) were co-transfected with1 ug vP35del viral DNA into Sf-21 cells by using Lipofectin fromGIBCO/BRL. Occlusion body formation was observed under light-microscopy3 days post-transfection. HEK293 cells were maintained in DMEM (IrvineScientific) supplemented with 10% FBS, 1 mM L-glutamine, andantibiotics. 293 cells (10⁶) were cotransfected by using Superfect™(Qiagen) with 0.1 ug of green fluorescence protein (GFP) marker plasmidpEGFP (CLONTECH), 0.25 ug of either pcDNA3-Bax or pcDNA3-Fas and 1.5 ugof pcDNA3-myc-BmIAP. Both floating and adherent cells were recovered 24to 36 hour post-transfection and pooled, and the percentage ofGFP-positive cells with nuclear apoptotic morphology was determined bystaining with 0.1 ug/ml 4′-6-diamidino-2-phenylindole (DAPI) (mean+/−S.D.; n=3) as described by Takhashi (1998) J. Biol. Chem.273:7787-7790.

The full-length BmIAP cDNA (SEQ ID NO:1) and the BmIAP polypeptide aminoacid sequence (SEQ ID NO:1) are: BmIAP cDNA nucleotide sequence: (SEQ IDNO:1)    1 CATTATTAAA CTCACTTCAC TTCGGTAGTG TGAATGTTAA CGTGAAACTCCGCGCTCTTC   61 TTTAGTTGCT ACTCGGTTCT GTCTGGCTGC GTTGACGTTT TGGAACTTCATACTATTTTG  121 TTCTTGCAAG ACGAGTGTCA GTGATTAAAC AAAAACATAA GAATAGACGTTTTATGCGTT  181 ACTAAAAAAA AGGAAAAATA TACCAATGGA GTTGACGAAA GTTGCTAAAAATGGAGCTGC  241 CGCCACGTTG GTGATGTTAA AAAATGCGCG GGATGCAAAA ATGCGACCTTTCATTGGTCC  301 GCTCATGTTA TCCTCGTGTG AGTCTTCAAC GACATCCACA CTCCCGTCACCTTCGTCGTC  361 AGCTGATAAA ACGGATAATC ACGACACATT CAACTTCCTT CCTGATATGCCCGACATGCG  421 TCGTGAAGAG GAACGTCTGA AAACATTTGA TCAGTGGCCC GTTACGTTTTTGACGCCGGA  481 ACAATTGGCC CGCAACGGAT TCTACTACCT CGGTCGCGGC GACGAAGTGTGCTGTGCTTT  541 CTGTAAGGTA GAAATTATGA GGTGGGTCGA AGGCGACGAT CCTGCCGCCGATCATCGGAG  601 ATGGGCGCCC CAGTGTCCCT TTGTACGAAA ACAAATGTAT GCCAACGCTGGGGGAGAGGC  661 GACCGCTGTC GGTAGAGACG AATGTGGGGC CAGTGCGGCC ACGCAGCCTCCCCGCATGCC  721 CGGCCCCGTG CACGCGCGGT ACTCCACCGA GGCCGCGCGG CTCGCCACCTTCAAGGACTG  781 GCCGAGACGT ATGCGCCAAA AACCCGAGGA ACTGGCAGAG GCCGGATTCTTCTATACAGG  841 CCAAGGTGAC AAAACGAAAT GCTTCTATTG CGACGGAGGG CTAAAAGATTGGGAAAGCGA  901 TGACGTTCCG TGGGAACAGC ACGCCAGATG GTTCGACCGC TGCGCGTACGTGCAATTGGT  961 GAAAGGACGT GACTACATTC AGAAGGTGAA GTCGGAGGCC ACTGCGATATCTGCTAGCGA 1021 AGAAGAACAG GCCGCCACCA ATGATTCGAC TAAGAACGTC GCCCAAGAGGGCGAGAAACA 1081 TTTGGATGAC TCTAAAATAT GTAAAATATG TTATTCCGAG GAGCGTAACGTGTGCTTCGT 1141 GCCGTGCGGC CACGTGGTGG CGTGCGCCAA GTGCGCGCTG TCGACGGACAAGTGCCCGAT 1201 GTGTCGCAGG ACGTTCACGA ATGCGGTGCG GCTCTACTTC TCGTGAAAGGACCCTCCTCG 1261 CGAGCTGTAT ACTAATCACT TCACCGGGCG GCCCTGGAGC GTGCTGAAACCACCCTTCGA 1321 ACGAAACCGC GTATCCTGTG ATTTTTACAT TAAATAAATT TACAAATTGATAGCGGTGGG 1381 GCAATGTATA GGAACTCGTC AGAACTCGCG AGTTGACGTG CAGGAAGGAGTTAGTGATTT 1441 GTAAACTTGT AAACTGATGT TGAAATGATT TTATTTATTA TTTAAAATTCTAATGACAAA 1501 GTGTAAGTAA ATAAATGTAC ATATTATTTT AGATTATCAG TTTGTCCCACCGACAAAAGT 1561 GAAATGTACA TAGGTGTTTT CATATCACTT CAACAGTCGA AGACCTTCTTTTTGAATTTA 1621 AGGATATATA TTTATACATA TAAATTAAAA TTTTAACGAG ACATCAATATAAATGGTTTA 1681 ACAACTTATT TATACACTGA AATCAAGTGA AGTGTAACAT GGTCTGAAGAATGTTTTACT 1741 GATTTCACTT CCCCTGTTGA AGTGATAAAA TTCTAATGTA AATCCAGAGTTTAAATGTCG 1801 TCATAATTAA TATAAGAAAC AAGTTTTACG CTTCTTTTGC TTGAAAAATCTTATAATTGA 1861 TTCAGGAATT ATTTAATGTG ACTATATTTT GTTCCTGTAA ATAACATAATATATACTATT 1921 TATTGATTAA TTCTGACATA ATTTATGGCA ATTCCGTAAG ATACAATCCAATACTTATTT 1981 CATGTAACTC ACTTCAAAAT AGTTGAATGT GTGGTGTGAT TATAATGTTAAATGTCTAAA 2041 TTTATAATAA ATTGAGCAAA GTTGCATTTA ATGTATGAAT ACTAATTATTGTTTTAACAA 2101 AACATTTAAG TATAATCTGC TCTGTGATTT TAATGTATCA AGAAATAACCCCAACACCTT 2161 AATTGAAGTT TTTACATTGT TGCTGATAAA AAAAATCATA TCAATTACATTTACAAGTCA 2221 ATTTTAATTG TTCAGAAACC AAACACAATT TTGTTAGTGA CTCCTGCTTTACGAAGTAGT 2281 ATGACAAACC AGTGTTTCGT TGATTGCATT AATTTAGTTG TAACCAATATTTACACTCAA 2341 CATTTTAAGA TGTCATTGAG GAATTCTGTA TAAAAAATGG GAATTTATTTATTGGTGTAT 2401 AATACAATCC CGCACAAGCC ATTTGCAAGT TTCTACACAA CTAAAACGTATTGTATCCAT 2461 TATCTATACG TCATATCATT AATATATACT TGCTTTAGCA AACATATATTCACGAATAAC 2521 TTCACAATAT ATTTTTGTAA ATCAACATAT TAATGGTAAT TAACGAATCGCACGGTACAA 2581 ATAGTGATAA CTGCTGAGTG CACTAAATAG TAAGAGAATT TATITAAACAGTCAAATTTT 2641 GTTTCATAAG TAGTTATTTC ATACTGTTGA ATGTTATTCA TTAAAACAAATGTTAAAGCA 2701 AAAAAAAAAA AAAAAAGTCG TGACTGGGAA AA BmIAP coding regionnucleotide sequence:    1 ATGGAGTTGA CGAAAGTTGC TAAAAATGGA GCTGCCGCCACGTTGGTGAT GTTAAAAAAT   61 GCGCGGGATG CAAAAATGCG ACCTTTCATT GGTCCGCTCATGTTATCCTC GTGTGAGTCT  121 TCAACGACAT CCACACTCCC GTCACCTTCG TCGTCAGCTGATAAAACGGA TAATCACGAC  181 ACATTCAACT TCCTTCCTGA TATGCCCGAC ATGCGTCGTGAAGAGGAACG TCTGAAAACA  241 TTTGATCAGT GGCCCGTTAC GTTTTTGACG CCGGAACAATTGGCCCGCAA CGGATTCTAC  301 TACCTCGGTC GCGGCGACGA AGTGTGCTGT GCTTTCTGTAAGGTAGAAAT TATGAGGTGG  361 GTCGAAGGCG ACGATCCTGC CGCCGATCAT CGGAGATGGGCGCCCCAGTG TCCCTTTGTA  421 CGAAAACAAA TGTATGCCAA CGCTGGGGGA GAGGCGACCGCTGTCGGTAG AGACGAATGT  481 GGGGCCAGTG CGGCCACGCA GCCTCCCCGC ATGCCCGGCCCCGTGCACGC GCGGTACTCC  541 ACCGAGGCCG CGCGGCTCGC CACCTTCAAG GACTGGCCGAGACGTATGCG CCAAAAACCC  601 GAGGAACTGG CAGAGGCCGG ATTCTTCTAT ACAGGCCAAGGTGACAAAAC GAAATGCTTC  661 TATTGCGACG GAGGGCTAAA AGATTGGGAA AGCGATGACGTTCCGTGGGA ACAGCACGCC  721 AGATGGTTCG ACCGCTGCGC GTACGTGCAA TTGGTGAAAGGACGTGACTA CATTCAGAAG  781 GTGAAGTCGG AGGCCACTGC GATATCTGCT AGCGAAGAAGAACAGGCCGC CACCAATGAT  841 TCGACTAAGA ACGTCGCCCA AGAGGGCGAG AAACATTTGGATGACTCTAA AATATGTAAA  901 ATATGTTATT CCGAGGAGCG TAACGTGTGC TTCGTGCCGTGCGGCCACGT GGTGGCGTGC  961 GCCAAGTGCG CGCTGTCGAC GGACAAGTGC CCGATGTGTCGCAGGACGTT CACGAATGCG 1021 GTGCGGCTCT ACTTCTCGTG A BmIAP polypeptideamino acid sequence: (SEQ ID NO:2)   1 MELTKVAKNGAAATLVMLKNARDAKMRPFI 31 GPLMLSSCESSTTSTLPSPSSSADKTDNHD  61 TFNFLPDMPDMRREEERLKTFDQWPVTFLT 91 PEQLARNGFYYLGRGDEVCCAFCKVEIMRW 121 VEGDDPAADHRRWAPQCPFVRKQMYANAGG151 EATAVGRDECGASAATQPPRMPGPVHARYS 181 TEAARLATFKDWPRRMRQKPEELAEAGFFY211 TGQGDKTKCFYCDGGLKDWESDDVPWEQHA 241 RWFDRCAYVQLVKGRDYIQKVKSEATAISA271 SEEEQAATNDSTKNVAQEGEKHLDDSKICK 301 ICYSEERNVCFVPCGHVVACAKCALSTDKC331 PMCRRTFTNAVRLYFS*

The full-length BmIAP cDNA (SEQ ID NO:1) (Genbank accession numberAF281073) contains a continuous open reading frame (ORF) encoding aprotein of 346 amino acids (FIG. 1B). This ORF is initiated by an AUGwithin a favorable context for translation (see, e.g., Kozak (1996)Mammalian Genomes 7:563-574) and is preceded by upstream stop codons inall three reading frames. FIG. 1A shows the location of the BIR domains(BIR1, residues 74 to 140; BIR2, residues 182 to 249; of SEQ ID NO:2)and the RING domain (residues 298 to 314; of SEQ ID NO:2) of BmIAP. FIG.1B is the full length amino acid sequence of BmIAP (SEQ ID NO:2).Sequence alignments of the BIR1 (FIG. 1C), BIR2 (FIG. 1D) and RING (FIG.1E) domains of BmIAP with the corresponding domains of other IAP familymembers are shown, with bold text indicates identical amino acid. TheGenbank accession numbers of sequences used for the alignments are:Bombyx mori IAP (BmIAP) AF281073, Spodoptera frugiperda IAP (SfIAP)AF186378, Trichoplusia ni IAP (TnIAP) AF195528, Orgyia pseudotsugatanucleopolyhedrovirus IAP (OpIAP) P41437, Cydia pomonella granulovirusIAP (CpIAP) P41436, and Drosophila melanogaster IAP1 (DIAP1) Q24306.

Similar to SfIAP, the BmIAP protein contains two BIR domains followed bya RING domain near its C-terminus (FIG. 1A and 1B). In the BIR domain ofBmIAP, the conserved presence and spacing of cysteine and histidineresidue (CX₂CX₆WX₉HX₆C) (SEQ ID NO:7) is also observed. Within BIR andRING regions, BmIAP shares 88% amino acid identity (92% similarity) withSfIAP, 90% identity (92% similarity) with TnIAP and 76% identity (81%similarity) with CpIAP (FIG. 1C-1E). Thus BmIAP shares high sequencesequence similarity with the other two lepidopteran IAPs, SfIAP andTnIAP, suggesting evolutionary conservation.

Experiments demonstrated that both BIR and RING domains of BmIAP arerequired to block apoptosis induced by apoptosis suppressorp35-deficient AcMNPV virus. The anti-apoptotic activity of BmIAP ininsect cells was tested by co-transfecting AcMNPV p35-deficient viralDNA into Sf-21 cells with plasmids containing full-length BmIAP (SEQ IDNO:2) or truncation mutants of BmIAP lacking two BIR domains or the RINGdomain (see FIG. 1A). Cells expressing IAPs/IAP fragments withanti-apoptotic activity support virus replication, whereas cells withouta functional IAP are unable to support virus replication and undergoapoptosis. Production of viable viral progeny from rescued virusesresults in the formation of occlusion bodies, serving as a convenientend-point. Occlusion body formation serves as a visual screeningend-point under light-microscopy, as described by Crook (1993) J. ofVirol. 67:2168-2174; Birnbaum (1994) J. of Virol. 68:2521-2525.Sf-21cells co-transfected with p35-deficient AcMNPV viral DNA and (FIG. 2A)BmIAP, or (FIG. 2D) SfIAP, show the production of occlusion bodies asindicated by arrowheads, whereas (FIG. 2B) BmIAP-BIR, (FIG. 2C)BmIAP-RING, and (FIG. 2E) AcIAP show apoptotic body formation (indicatedby arrowheads) without occlusion body formation. Since SfIAP has beenshown to block apoptosis in both insect and mammalian cells (see, e.g.,Huang (2000) supra), whereas AcIAP is ineffective (Clem (1994) Mol.Cell. Biol. 14:5212-5222; Huang (2000) supra, SfIAP and AcIAP were usedas positive and negative controls, respectively (FIG. 2D and E). FIG. 2Fis the mock transfection control.

A plasmid encoding the full-length BmIAP (SEQ ID NO:2) was able tocomplement the p35 (apoptosis inhibiting)-deficiency in the baculovirus,supporting occlusion body production and virus replication at 3 dayspost-transfection (FIG. 2A). In contrast, neither the BIR nor RINGdomain deletion mutants of BmIAP (see FIG. 1A) was able to supportocclusion body formation (thus, no apoptosis inhibiting activity). Sf-21cells displayed morphological changes of apoptosis, such as apoptoticbody formation, when either the BIR or RING domain deletion mutants wereco-transfected with p35-deficient viral DNA (FIG. 2B and C). Theseresults demonstrate that both the BIR and RING domain regions of BmIAP(BIR1, residues 74 to 140; BIR2, residues 182 to 249; of SEQ ID NO:2)and the RING domain (residues 298 to 314; of SEQ ID NO:2) are requiredin combination for the anti-apoptotic function in insect cells.

Experiments demonstrated that BmIAP inhibited Bax-induced but notFas-induced apoptosis in mammalian cells (i.e., BmIAP protects mammaliancells against Bax-induced but not Fas-induced apoptosis). Expressionplasmid encoding Bax (FIG. 3A) or Fas (FIG. 3B) were co-transfected intoHEK 293 cells with the indicated myc-tagged IAP expression plasmids.Percentage apoptosis was measured 24 to 36 hours post-transfection by4′-6-diamidino-2-phenylindole (DAPI) staining (mean +/−S.D., n=3).Recombinant BmIAP (2 uM) was added to cytosolic extracts (10 mg/ml) fromHEK293 cells concurrently with the addition of 1 uM cytochrome-c/10 mMdATP. After incubation at 30° C. for 10 minutes, aliquots were withdrawnand assayed for caspase activity, as measured by release of AFC fromAc-DEVD-AFC substrate (100 uM). Data are presented in FIG. 3C as apercentage relative to control reaction in which cytochrome-c/dATP wereadded alone.

SfIAP and baculoviral IAPs were previously shown to block apoptosis inmammalian cells (Huang (2000) supra; Hawkins (1996) Proc. Natl. Acad.Sci. USA 93:13786-13790; Uren (1996) Proc. Natl. Acad. Sci. USA93:4974-4978; Hawkins (1998) Cell Death and Differentiation 5:569-576).To explore whether BmIAP has similar properties, we co-expressed BmIAPin HEK293 cells with either Fas or Bax, representing two major pathwaysthat utilize caspase-8 and caspase-9, respectively, as their apicalproteases. Similar to SfIAP, full-length BmIAP (SEQ ID NO:2) inhibitedBax (FIG. 3A) but not Fas-induced apoptosis (FIG. 3B). In contrast humanXIAP protected cells against both Bax and Fas-induced apoptosis. As inSf-21 cells, the inhibition of Bax-induced apoptosis in mammalian cellsalso requires both the BIR and RING domains of BmIAP (SEQ ID NO:2) (seeFIG. 1A), suggesting the conservation of the structural requirements forinhibition (FIG. 3A). Immunoblot analysis indicated that the levels ofthe BIR and RING truncation proteins were similar to that of full-lengthBmIAP in transfected cells, excluding differences in protein levels asan explanation for the failure of the BIR domains or RING domain tosuppress cell death.

These results were further confirmed in a cell-free system in whichexogenously added cytochrome-c, an agonist of the caspase-9 activatingprotein Apaf-1 (see, e.g., Zou (1997) Cell 90:405-413), inducedactivation of caspase-3 and similar effector proteases. BmIAP directlysuppressed caspase-9, but not caspase-3 or caspase-7. This was measuredby hydrolysis of Ac-DEVD-AFC, as described by Quan (1995) J. Biol. Chem.270:10377-10379). Recombinant active (FIG. 4A) caspase-9 was added at0.2 uM and incubated at 37° C. with Ac-LEHD-AFC substrate (100 uM) inthe presence or absence of various concentration (0.2-1.6 uM) ofrecombinant purified BmIAP or SfIAP. AFC release was measuredcontinuously. In FIG. 4, data are expressed as a percentage relative tocontrol reactions lacking IAPs, using rates determined from the linearportion of enzyme progress curves. Various control GST-fusion proteinshad no inhibition effect.

Recombinant caspase-3 (2.6 nM) was incubated at 37° C. with Ac-DEVD-AFCsubstrate (100 uM) in the presence or absence of 0.05 uM GST-XIAP, 0.5uM GST-BmIAP (200 fold molar excess to caspase) or 0.5 uM GST-SfIAP (200molar excess) (FIG. 4B). AFC release was measured as above.

Recombinant caspase-7 (7.0 nM) was incubated at 37° C. with Ac-DEVD-AFCsubstrate (100 uM) in the presence or absence of 0.14 uM GST-XIAP, 0.7uM GST-BmIAP (100 fold molar excess relative to caspase or 0.7 uMGST-SfIAP (100 molar excess) (FIG. 4C). AFC release was measured asabove. In cytosolic extracts treated with cytochrome-c, recombinantBmIAP and positive control recombinant SfIAP completely blocked thehydrolysis of Ac-DEVD-AFC whereas negative control recombinant XIAP-BIR1had no effect on caspase activity (FIG. 4C). Since caspases-3 and -7 arecommon to both Bax and Fas pathways, these results demonstrate thatBmIAP, like SfIAP, inhibits the mitochondria/cytochrome-c pathway inmammalian cells, thus, suppressing apoptosis at a step upstream ofcaspases-3 and -7. This finding is supported by the observation thatBmIAP does not inhibit caspases-3 and -7 in vitro.

These experiments demonstrated that BmIAP is a direct inhibitor ofcaspase-9. Purified recombinant BmIAP was incubated with purifiedrecombinant caspase-9. Residual activity was measured using Ac-LEHD-AFCas a substrate of caspase-9. BmIAP inhibited recombinant caspase-9 in aconcentration-dependent manner. The relative amount of BmIAP requiredfor caspase-9 inhibition was about 8 fold molar excess (FIG. 4A),similar to the results reported previous for SfIAP and XIAP (Deveraux(1999) supra; Huang (2000) supra; SfIAP was shown to directly inhibitcaspase-9). Unlike XIAP, but similar to SfIAP, BmIAP did not inhibitrecombinant caspase-3 and caspase-7 (caspases-3, -7 and -9 are involvedin apoptotic pathway induced by Bax), suggesting a narrower range ofcaspases specificity compared to human XIAP (FIG. 4B and C).

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1-19. (canceled)
 20. A non-human transgenic animal comprising a nucleicacid sequence having at least 95% sequence identity to SEQ ID NO:1. 21.The nonhuman transgenic animal of claim 20, wherein the animal is a rator a mouse.
 22. The nonhuman transgenic animal of claim 20, wherein thenucleic acid encodes a polypeptide having a sequence as set forth in SEQID NO:2.
 23. The nonhuman transgenic animal of claim 20, wherein thenucleic acid encodes a polypeptide capable of inhibiting apoptosis. 24.A transgenic plant comprising a nucleic acid sequence having at least95% sequence identity to SEQ ID NO:1.
 25. The transgenic plant of claim24, wherein the nucleic acid encodes a polypeptide capable of inhibitingapoptosis.
 26. The transgenic plant of claim 24, wherein the plant isabiotic or biotic insult resistant.
 27. The transgenic plant of claim26, wherein the biotic insult is induced by a plant pathogen.
 28. Thetransgenic plant of claim 27, wherein the plant pathogen is a virus, afungus, a bacteria or a nemotode.
 29. The transgenic plant of claim 26,wherein the abiotic insult is induced by high moisture, low moisture,salinity, nutrient deficiency, air pollution, high temperature, lowtemperature, soil toxicity, herbicides or insecticides.
 30. Thetransgenic plant of claim 24, wherein at least a portion of the plantexhibits a decreased level of senescence.
 31. A seed capable ofgerminating into a plant having in its genome a heterologous nucleicacid sequence having at least 95% sequence identity to SEQ ID NO:1. 32.The seed of claim 31, wherein the nucleic acid encodes a polypeptidecapable of inhibiting apoptosis in a plant cell.
 33. An isolated orrecombinant polypeptide comprising a sequence having at least 95%sequence identity to SEQ ID NO:2.
 34. The isolated or recombinantpolypeptide of claim 33, wherein the polypeptide is capable ofinhibiting apoptosis in insect cells.
 35. The isolated or recombinantpolypeptide of claim 33, wherein the polypeptide is capable ofinhibiting apoptosis in Bombyx mori cells.
 36. The isolated orrecombinant polypeptide of claim 33, wherein the polypeptide is capableof inhibiting apoptosis in mammalian cells.
 37. The isolated orrecombinant polypeptide of claim 33, wherein the nucleic acid encodes apolypeptide capable of inhibiting apoptosis in plant cells.
 38. Theisolated or recombinant polypeptide of claim 33, wherein the polypeptideis capable of inhibiting caspase
 9. 39. An isolated or recombinantpolypeptide comprising a sequence as set forth in SEQ ID NO:2.
 40. Afusion protein comprising a sequence having at least 95% sequenceidentity to SEQ ID NO:2 and a second domain.
 41. The fusion protein ofclaim 40, wherein the second domain comprises glutathione S-transferase(GST).
 42. An antibody or binding fragment thereof, wherein the antibodyor fragment specifically binds to a polypeptide or an immunogenicfragment thereof, wherein the polypeptide comprises a sequence having atleast 95% sequence identity to SEQ ID NO:2.
 43. An antibody or bindingfragment thereof, wherein the antibody or fragment specifically binds toa protein having an amino acid sequence as set forth in SEQ ID NO:2 oran immunogenic fragment thereof.
 44. (canceled)
 45. A method ofdetecting or isolating a polypeptide, wherein the polypeptide comprisesa sequence having at least 95% sequence identity to SEQ ID NO:2,comprising contacting a biological sample with an antibody as set forthin claim 42 or claim
 43. 46. (canceled)
 47. A method for inhibitingapoptosis in a cell comprising the following steps: (a) providing anisolated or recombinant polypeptide comprising a sequence having atleast 95% sequence identity to SEQ ID NO:2, wherein the polypeptide iscapable inhibiting apoptosis in the cell, and (a) contacting thepolypeptide with the cell in an amount sufficient to inhibit apoptosisin the cell.
 48. A method for inhibiting apoptosis in a cell comprisingthe following steps: (a) providing an isolated or recombinant nucleicacid comprising a sequence having at least 95% sequence identity to SEQID NO:1, wherein the nucleic acid encodes polypeptide capable ofinhibiting apoptosis in the cell, and (b) contacting the nucleic acidwith the cell and expressing the nucleic acid to produce an amount ofpolypeptide sufficient to inhibit apoptosis in the cell.
 49. The methodof claim 47 or claim 48, wherein the cell is an insect cell.
 50. Themethod of claim 49, wherein the insect cell is a Bombyx mori cell. 51.The method of claim 49, wherein the insect cell is a Spodopterafrugiperda cell.
 52. The method of claim 47 or claim 48, wherein thecell is a mammalian cell.
 53. The method of claim 47 or claim 48,wherein the cell is a plant cell.
 54. A method for identifying an agentthat can modulate the activity of a polypeptide, wherein the polypeptidecomprises a sequence having at least 95% sequence identity to SEQ IDNO:2 and is capable inhibiting a caspase 9 protease, comprising: (a)providing an isolated or recombinant polypeptide comprising a sequencehaving at least 95% sequence identity to SEQ ID NO:2 that is capableinhibiting a caspase 9 protease, and a test agent, (b) contacting thecaspase 9 protease and polypeptide in the presence and absence of thetest agent; and (c) measuring the ability of the polypeptide to inhibitthe caspase 9 protease in the presence and absence of the test agent,wherein an increase or decrease in the ability of the polypeptide toinhibit the caspase 9 protease in the presence of the test agentidentifies the test agent as a modulator of the polypeptide's activity.55. A method for identifying an agent that can modulate the activity ofa polypeptide, wherein the polypeptide comprises a sequence having atleast 95% sequence identity to SEQ ID NO:2 and is capable inhibitingapoptosis in a cell, comprising: (a) contacting a cell expressing thepolypeptide recombinantly in the presence and absence of a test agentbefore, during or after inducing apoptosis in the cell; and (b)measuring the amount or degree of polypeptide activity in the cell inthe presence and absence of the test agent, wherein an increase ordecrease in the amount or degree of polypeptide activity in the cell inthe presence of the test agent identifies the test agent as a modulatorof the polypeptide's activity.
 56. The method of claim 55, wherein thecell is an insect cell.
 57. The method of claim 56, wherein the cell isa Bombyx mori cell.
 58. The method of claim 55, wherein the cell is aplant cell.
 59. The method of claim 55, wherein the cell is a mammaliancell.
 60. The method of claim 55, wherein the cell is a yeast cell. 61.The method of claim 55, wherein the degree of polypeptide activity inthe cell is determined by measuring the amount or degree of apoptosis inthe cell.
 62. The method of claim 55, wherein the degree of polypeptideactivity in the cell is determined by measuring the amount or degree ofcaspase protease activity in the cell.
 63. The method of claim 55,wherein the degree of polypeptide activity in the cell is determined bymeasuring the amount or degree of DNA fragmentation in the cell.
 64. Themethod of claim 55, wherein the degree of polypeptide activity in thecell is determined by measuring the amount or degree of cleavage ofsubstrates of caspases in the cell.
 65. A method of generating anabiotic or biotic insult-resistant plant comprising the following steps(a) providing an isolated or recombinant polypeptide comprising asequence having at least 95% sequence identity to SEQ ID NO:2, whereinthe polypeptide is capable inhibiting apoptosis in a plant cell, and (a)contacting the polypeptide with the plant in an amount sufficient toinhibit apoptosis in the plant, thereby generating a plant that isbiotic insult resistant.
 66. A method for generating an abiotic orbiotic insult-resistant plant comprising the following steps: (a)providing an isolated or recombinant nucleic acid comprising a sequencehaving at least 95% sequence identity to SEQ ID NO:1, wherein thenucleic acid encodes polypeptide capable of inhibiting apoptosis in aplant cell, and (b) contacting the nucleic acid with the plant andexpressing the nucleic acid to produce an amount of polypeptidesufficient to inhibit apoptosis in the plant.
 67. The method of claim 65or claim 66, wherein the biotic insult is induced by a plant pathogen.68. The method of claim 67, wherein the plant pathogen is a virus, afungus, a bacteria or a nemotode.
 69. The method of claim 65 or claim66, wherein the abiotic insult is induced by high moisture, lowmoisture, salinity, nutrient deficiency, air pollution, hightemperature, low temperature, soil toxicity, herbicides or insecticides.